ABSTRACT Despite exosome (Ex) tumor specificity, high stability in easily assessable biological fluids such as blood and reflection of same heterogeneity as tumors themselves, the use of Ex for cancer biomarkers has been challenging due to lack of gold standards or ideal techniques to reliably isolate and precisely determine the biophysical and bio-molecular characteristics of isolated Ex populations. Increasing evidence that variations in Ex isolation strategies significantly impact downstream analysis warrants quantitative, high resolution evaluation of Ex isolates from different methods at the single vesicle level and to assess the efficacy, rigor, and reproducibility of Ex isolates. To address the current lack of understanding of the inherent methods-dependent variations in Ex, the current proposal brings together high-resolution PeakForce (PF)TM imaging with correlative proteomics using Western blot, Single Molecule Force Spectroscopy (SMFS) & Mass spectrometry (MS) to assess the efficacy, rigor, and reproducibility of cancer cell-type and method-dependent Ex isolates in well established breast cancer cell model. We propose to develop Exonanometrics method- based on the biophysical and bio-molecular characteristics of Ex, to test the comparative performance of Ex isolation techniques and facilitate selection of isolation method/s for pre-specified Ex based cancer biomarker applications. Aim 1 will compare biophysical metrics of Ex isolates based on quantitative, high resolution, PFTM imaging of in vitro breast cancer cells derived Ex, obtained by different isolation methods. Aim 2 will compare bio-molecular metrics in Ex isolates by probing surface protein composition of breast cancer cell derived Ex via Western blots, SMFS, and MS proteomic analysis. Descriptive statistics and mixed effects regression models to integrate biophysical and proteomic characteristics obtained from nanoscale PFTM imaging, SMFS and MS proteomics will be used to develop Exonanometrics. We envisage that successful completion of the proposed study will provide fundamental knowledge of structure, bio-molecular characteristics of Ex populations obtained from different methods at the single vesicle level with molecular resolution. Exonanometrics will establish first nanoscale analytical standards for the Ex community, to chose the best Ex isolation strategy based on prospective downstream cancer diagnostic assays.